An important step in the life cycle of viruses is assembly of the virion (virus particle). For most retroviruses assembly takes place at the plasma membrane, where viral proteins and the RNA genome come together to form a budding nascent virion, which eventually becomes enveloped by membrane, pinching off from the cell. The long term goals of the proposed research are to further elucidate the process of retrovirus assembly, using the avian Rous sarcoma virus as a model system. The experiments, which emphasize biochemical approaches, are grouped under four specific aims. 1. Protein-RNA and protein-protein interactions. The experiments are designed to: (a) elucidate the role of the Gag NC domain, and of foreign protein interaction modules that can replace it, in assembly; (b) develop strategies for incorporating functional protease, reverse transcriptase, and integrase proteins into particles assembled in vitro; (c) further define the packaging sequence psi and how the NC domain interacts with psi, by selection of infectious viruses from a pool of proviruses with locally randomized sequences; (d) explore Gag-Gag interactions in living cells using fluorescence resonance energy transfer with GagGFP. 2. Protein-membrane interactions in assembly. The proposed experiments will: (a) define the parameters and biological relevance for Gag and MA binding to liposomes, and explore methods for reconstituting a membrane around immature viral cores or particles assembled in vitro; (b) determine the origin of the lipid raft-like composition of viral membranes, and with collaborators define the properties of GagGFP budding sites on the membrane, using single particle tracking in real time. 3. Late functions in assembly. The proposed experiments will: (a) identify late domain binding proteins and enzymes involved in ubiquitin metabolism that are incorporated into virions; (b) explore permeabilized cells as a model for studying the late steps in assembly. 4. Three dimensional structures of Gag proteins. Our structural biology collaborators will seek high resolution structures for: (a) CA and extended immature versions of CA; (b) the C-terminal CA domain extended through NC; (c) the portion of Gag including the late domain PPPY; (d) the monomeric PR domain as part of Gag. For all of these studies the principles that are learned should be applicable to HIV and to other retroviruses.